The mapping and manipulation of complex genomes will be greatly facilitated by the availability of reagents that cleave DNA at sites that occur infrequently. Such reagents will also be useful for gene targeting projects that am to correct the inborn mutations that cause genetic disease. A family of related DNA endonucleases has been identified that will accelerate these studies. A common feature of these enzymes is that they initiate genomic rearrangement events within their host organisms by making double-stranded breaks at specific sites. The molecular mechanism of these enzymes is unknown. However, the conservation of structural features in this family, including the presence of two dodecapeptide motifs, suggests that information learned about the structure and function of one enzyme will be directly applicable to the other members of the family. The objective of the proposed research is to understand how the structural and mechanistic features of one such endonuclease from yeast, called VDE, results in site-specific cleavage. The VDE endonuclease has been purified to homogeneity as an active enzyme and its substrate has been identified. The first aim is to determine whether VDE cleaves DNA by a concerted or a sequential reaction mechanism and to ascertain the oligomeric form of the enzyme during cleavage. Kinetic parameters for VDE-mediated cleavage will be measured under a variety of conditions using cognate and non-cognate substrates to reveal the reaction mechanism. The oligomeric state of the enzyme will be deduced from gel-shift experiments. The second goal is to assess the relative importance of the base-pairs within the recognition site in the DNA cleavage reaction. The recognition site will be randomly mutagenized, and these substrates will be tested for VDE binding and cleavage activities. An optimized cleavage/recognition site will be generated by a novel PCR-based technique. The third aim is to determine which amino acids within the dodecapeptide motifs and elsewhere in the protein affect catalytic function and determine specificity. Mutant proteins generated by site-directed and cassette mutagenesis will be assayed for catalytic and DNA-binding function and the mutations will be located by sequence analysis. Chimeric proteins will be constructed between VDE and the related HO endonuclease in order to localize the specificity determinants. In the long term we would like to use this data to alter substrate specificity.